Identification and characterization of carbonylation sites in trastuzumab biosimilars.

Detection of metal catalyzed carbonylation in proteins is traditionally based on derivatization followed by detection and quantification via spectroscopy or immunodetection. However, these measure only cumulative carbonylation and do not identify the specific sites of modification within the protein. Recently, fluorescein thiosemicarbazide (FTC) based semi-microplate method was adapted for high throughput monitoring of carbonyl content during mAb process development, using size-exclusion chromatography followed by ultraviolet and fluorescence detection. Here, we have examined carbonylation in originators and 4 biosimilars of an IgG1 therapeutic monoclonal antibody, trastuzumab, a first line of therapy for HER2 positive breast cancer. The hyphenated RP-ESI-MS/MS detection was able to identify the location of each of the carbonylated amino acids for all products. The result is a comprehensive map of a total of 27 unique identified carbonylation sites of trastuzumab found across multiple batches of originator as well as marketed biosimilars. Our results demonstrate that although the different carbonylation sites are spread across different domains throughout the mAb sequence, the complementarity determining regions (CDRs) are free of carbonylation and all identified sites lie within the framework region of the variable domain. Moreover, the constant- heavy domain 3 (CH3) region seems to be particularly resistant to process induced carbonylation.

Alterations in Synaptic Plasticity and Oxidative Stress Following Long-Term Paracetamol Treatment in Rat Brain.

Several studies have recently revealed that cognitive function can be affected by paracetamol (APAP) treatment. However, the exact impact of this drug treatment on learning and memory has not been clarified. This study aimed to investigate the effect of APAP treatment on the alteration of synapses and oxidative stress in the rat frontal cortex and hippocampus. APAP at a dose of 200 mg/kg bw was fed to adult male Wistar rats through either acute (n = 10), 15-day (n = 10), or 30-day (n = 10) treatment regimens. The synaptic ultrastructure and proteins, synaptophysin (SYP) and postsynaptic density-95 (PSD-95), were monitored. The amount of protein carbonyl oxidation (PCO) and glutathione (GSH) levels were examined. Our results demonstrated that acute treatment with APAP had no effect on synapses and oxidative stress. However, the synapses obtained from rats with 15-day APAP treatment showed a marked shortening of active zones and widening of the synaptic cleft. Decrement of SYP and PSD-95 proteins were demonstrated in these rats as well. With 30-day APAP treatment, the alteration of the synaptic ultrastructure and proteins was more evident. Moreover, the depletion of GSH and the elevation of PCO levels were demonstrated in the rats treated with APAP for 30 days. These results suggest that long-term APAP treatment can induce synaptic degeneration in the hippocampus and frontal cortex. The increase in oxidative stress in these brain areas may be due to the deleterious effect of this drug.

The effect of short-term methionine restriction on glutathione synthetic capacity and antioxidant responses at the whole tissue and mitochondrial level in the rat liver.

Dietary methionine restriction (MR) where methionine is the sole source of sulfur amino acid increases lifespan in diverse species. Methionine restricted rodents experience a decrease in glutathione (GSH), a major antioxidant, in several tissues, which is paradoxical to longevity interventions because tissues with low GSH might experience more oxidative damage. Liver plays a key role in GSH synthesis and here we examined how MR influences GSH metabolism in the liver. We also hypothesised that low GSH might be subsidized by compensatory pathway(s) in the liver. To investigate GSH synthesis and antioxidant responses, Fischer-344 rats were given either a MR diet or a control diet for 8 weeks. Based on γ-glutamylcysteine synthetase activity, GSH synthetic capacity did not respond to low dietary methionine availability. Tissue level protein and lipid oxidation markers do not support elevated oxidative damage, despite low GSH availability. Whole tissue and mitochondrial level responses to MR differed. Specifically, the activity of glutathione reductase and thioredoxin reductase increase in whole liver tissue which might offset the effects of declined GSH availability whereas mitochondrial GSH levels were unperturbed by MR. Moreover, enhanced proton leak in liver mitochondria by MR (4 week) presumably diminishes ROS production. Taken together, we suggest that the effect of low GSH in liver tissue is subsidized, at least in part, by increased antioxidant activity and possibly by enhanced mitochondrial proton leak.

Inspiratory threshold loading reduces lipoperoxidation in obese and normal-weight subjects.

Obesity is related to increased oxidative stress. Although low-intensity physical exercise reduces oxidative stress, obese subjects may show exercise intolerance. For these subjects, inspiratory threshold loading could be an alternative tool to reduce oxidative stress. We investigated the effects of inspiratory threshold loading on biomarkers of oxidative stress in obese and normal-weight subjects. Twenty obese (31.4 ± 6 years old, 10 men and 10 women, 37.5 ± 4.7 kg/m2) and 20 normal-weight (29.4 ± 8 years old, 10 men and 10 women, 23.2 ± 1.5 kg/m2) subjects matched for age and gender participated in the study. Maximal inspiratory pressure (MIP) was assessed by a pressure transducer. Blood sampling was performed before and after loading and control protocols to assess thiobarbituric acid reactive substances (TBARS), protein carbonylation, and reduced glutathione. Inspiratory threshold loading was performed at 60% MIP and maintained until task failure. The 30-min control protocol was performed at 0 cmH2O. Our results demonstrated that inspiratory threshold loading reduced TBARS across time in obese (6.21 ± 2.03 to 4.91 ± 2.14 nmol MDA/ml) and normal-weight subjects (5.60 ± 3.58 to 4.69 ± 2.80 nmol MDA/ml; p = 0.007), but no change was observed in protein carbonyls and glutathione in both groups. The control protocol showed no significant changes in TBARS and protein carbonyls. However, reduced glutathione was increased across time in both groups (obese: from 0.50 ± 0.37 to 0.56 ± 0.35 µmol GSH/ml; normal-weight: from 0.61 ± 0.11 to 0.81 ± 0.23 µmol GSH/ml; p = 0.002). These findings suggest that inspiratory threshold loading could be potentially used as an alternative tool to reduce oxidative stress in both normal-weight and obese individuals.

Ecobiological approach to research regarding ageing and diseases.

Ageing and age-related diseases (ARD) share a common biological clock that appears as their root cause: protein damage. A majority of proteins have evolved into native structures resistant to oxidative damage but any folding imperfections, including those due to "silent" amino acid substitutions, reduce oxidation resistance. Damaged proteins accumulate with age and trigger ageing-like phenotypes reversible by their turnover, while acquired genome alterations remain as stable consequences of protein malfunction. Ageing and ARD display species-specific latency in phenotypic expression. Disease latency may be proposed as to be due to phenotypic suppression of cellular defects by molecular traffic among neighbouring cells. Such cross-complementation of functional deficiencies acts as a kind of tissue-based cellular "solidarity", called cellular parabiosis. Chronic inflammation reveals dormant cell phenotypes and shortens disease latency by the breakdown of cell-cell communication, as in tumour promotion and inflammation. At the present time, predictive diagnostics, prognostics, prevention and even cure of disease by phenotypic reversion become conceivable.

Redox Profiling Reveals Clear Differences between Molecular Patterns of Wound Fluids from Acute and Chronic Wounds.

Wound healing is a complex multiphase process which can be hampered by many factors including impaired local circulation, hypoxia, infection, malnutrition, immunosuppression, and metabolic dysregulation in diabetes. Redox dysregulation is a common feature of many skin diseases demonstrated by virtually all cell types in the skin with overproduction of reactive oxygen and nitrogen species. The objective of this study was to characterize the redox environment in wound fluids and sera from patients suffering from chronic leg ulcers (n = 19) and acute wounds (bulla fluids from second degree burns; n = 11) with serum data also compared to those from healthy volunteers (n = 7). Significantly higher concentrations of TNF-α, interleukine-8, vascular endothelial growth factor, and lactate dehydrogenase (measure of cell damage) were found in fluids from chronic wounds compared to acute ones. The extent of protein carbonylation (measure of protein oxidation), lipid peroxidation, and tyrosine nitration (indicator of peroxynitrite production) was similar in acute and chronic wound fluids, while radical scavenging activity and glutathione (GSH) levels were elevated in chronic wound fluids compared to acute wounds. Sera were also assessed for the same set of parameters with no significant differences detected. Nitrotyrosine (the footprint of the potent oxidant peroxynitrite) and poly(ADP-ribose) (the product of the DNA damage sensor enzyme PARP-1) could be detected in wound biopsies. Our data identify multiple signs of redox stress in chronic wounds with notable differences. In chronic wounds, elevations in antioxidant levels/activities may indicate compensatory mechanisms against inflammation. The presence of nitrotyrosine and poly(ADP-ribose) in tissues from venous leg ulcers indicate peroxynitrite production and PARP activation in chronic wounds.

The in vivo and in vitro approaches for establishing a link between advanced glycation end products and lung cancer.

Advanced glycation end products (AGEs) are directly related to third aging-associated diseases, such as cardiovascular diseases, arteriosclerosis, and neurodegeneration. Likewise, these irreversible and nonenzymatic products have been reported to be involved in the progression of malignant cancers. In general, aging-associated diseases and the initiation of cancer have been subjects of interest for several years. Few studies on the role of AGEs in cancer have been performed on cell lines. Moreover, past investigations in the field of glycation biology still lack the knowledge of in vivo and in vitro approaches for cancer cells. Accordingly, we aimed to focus on and establish a link between cancer and glycation with respect to all the possible AGEs. In our study, the levels of carboxymethyllysine (CML) increased by 50.94% in an animal model of glycation, whereas in an animal model of cancer, the contents of CML increased by 45.94% compared with their negative controls. Similarly, fluorescent AGEs were also examined and were found to be increased by 65.3% and 58.63% in the animal models of glycation and cancer, respectively, compared with the control subjects. The protein carbonyl contents were also found to be enhanced in the animal models of glycation and cancer. In our study, the levels of reactive oxygen species were also found to be significantly increased in the in vitro model of cancer cells as compared with the controls. Such an initial breakthrough indicated that AGEs were present in the serum of the animal models of cancer and glycation.

Effect of oxidative stress on vital indicators of Acanthamoeba castellanii (T4 genotype).

Acanthamoeba has 22 genotypes with the T4 genotype being the main causative agent of amoebic granulomatous encephalitis and keratitis. Because the molecular mechanisms of the immune defenses of neutrophils and macrophages against histoparasites are based on oxidative stress, parasites may rely on their antioxidant systems to preclude immune defenses. Therefore, understanding of the effect of oxidative stress on vital characteristics of Acanthamoeba castellanii (T4 genotype) and the antioxidant defense responses of Acanthamoeba to oxidative status will cast light on immune cell-parasite interactions. Acanthamoeba T4 cells were cultured in RPMI-1640 medium containing different concentrations of hydrogen peroxide (H2O2). The survival of Acanthamoeba was evaluated by MTT assay and the IC50 concentration was calculated. The total antioxidant capacity (TAC) of the parasite was determined by the cupric reducing antioxidant capacity (CUPRAC) method. Malondialdehyde (MDA) as a marker of lipid peroxidation, protein carbonyl content as a measure of oxidized protein, total thiol (-SH) groups present on proteins as a major source of cellular antioxidants, and total oxidant status (TOS) were evaluated by colorimetric methods. The reactive oxygen species level increased markedly after induction of oxidative stress by the treatment of Acanthamoeba T4 with H2O2. Exposure to H2O2 also significantly increased the MDA and protein carbonyl content. The TOS level and total thiol groups also increased in the treated group compared to those in untreated parasites, although the results were not statistically significant. The TAC level was found to be significantly higher in H2O2-treated parasites, confirming that the parasite fosters its total antioxidant capacity to overcome oxidative conditions. This study showed that under oxidative stress, the defense reactions of the parasite are in part mediated by increasing its antioxidant activity, which is important for the survival of the parasite.

Knockout of the Gsta4 Gene in Male Mice Leads to an Altered Pattern of Hepatic Protein Carbonylation and Enhanced Inflammation Following Chronic Consumption of an Ethanol Diet.

BACKGROUND: Glutathione S-transferase A4-4 (GSTA4) is a key enzyme for removal of toxic lipid peroxidation products such as 4-hydroxynonenal (4-HNE). In this study, we examined the potential role of GSTA4 on protein carbonylation and progression of alcoholic liver disease by examining the development of liver injury in male wild-type (WT) SV/J mice and SV/J mice lacking functional GSTA4 (GSTA4-/- mice). METHODS: Adult male WT and GSTA4-/- mice were fed chow (N = 10 to 12) or high-fat Lieber-DeCarli liquid diets containing up to 28% calories as ethanol (EtOH) (N = 18 to 20) for 116 days. At the end of the study, half of the EtOH-fed mice were acutely challenged with an EtOH binge (3 g/kg given intragastrically) 12 hours before sacrifice. Carbonylation of liver proteins was assessed by immunohistochemical staining for 4-HNE adduction and by comprehensive liquid chromatography-tandem mass spectrometry (LC-MS/MS) of purified carbonylated proteins. RESULTS: Chronic EtOH intake significantly increased hepatic 4-HNE adduction and protein carbonylation, including carbonylation of ribosomal proteins. EtOH intake also resulted in steatosis and increased serum alanine aminotransferase. Hepatic infiltration with B cells, T cells, and neutrophils and mRNA expression of pro-inflammatory cytokines tumor necrosis factor (TNF)α and interferon (IFN)γ was modest in WT mice. However, an EtOH binge increased hepatic necrosis, hepatic cell proliferation, and expression of TNFα mRNA (p < 0.05). EtOH treatment of GSTA4-/- mice increased B-cell infiltration and increased mRNA expression of TNFα and IFNγ and of matrix remodeling markers MMP9, MMP13, and Col1A1 (p < 0.05). GSTA4-/- mice exhibited panlobular rather than periportal distribution of 4-HNE-adducted proteins and increased overall 4-HNE staining after EtOH binge. Comprehensive LC-MS of carbonylated proteins identified 1,022 proteins of which 189 were unique to the GSTA4-/- group. CONCLUSIONS: These data suggest long-term adaptation to EtOH in WT mice does not occur in GSTA4-/- mice. Products of lipid peroxidation appear to play a role in inflammatory responses due to EtOH. And EtOH effects on B-cell infiltration and autoimmune responses may be secondary to formation of carbonyl adducts.

Early alpha-lipoic acid therapy protects from degeneration of the inner retinal layers and vision loss in an experimental autoimmune encephalomyelitis-optic neuritis model.

BACKGROUND: In multiple sclerosis (MS), neurodegeneration is the main reason for chronic disability. Alpha-lipoic acid (LA) is a naturally occurring antioxidant which has recently been demonstrated to reduce the rate of brain atrophy in progressive MS. However, it remains uncertain if it is also beneficial in the early, more inflammatory-driven phases. As clinical studies are costly and time consuming, optic neuritis (ON) is often used for investigating neuroprotective or regenerative therapeutics. We aimed to investigate the prospect for success of a clinical ON trial using an experimental autoimmune encephalomyelitis-optic neuritis (EAE-ON) model with visual system readouts adaptable to a clinical ON trial. METHODS: Using an in vitro cell culture model for endogenous oxidative stress, we compared the neuroprotective capacity of racemic LA with the R/S-enantiomers and its reduced form. In vivo, we analyzed retinal neurodegeneration using optical coherence tomography (OCT) and the visual function by optokinetic response (OKR) in MOG35-55-induced EAE-ON in C57BL/6J mice. Ganglion cell counts, inflammation, and demyelination were assessed by immunohistological staining of retinae and optic nerves. RESULTS: All forms of LA provided equal neuroprotective capacities in vitro. In EAE-ON, prophylactic LA therapy attenuated the clinical EAE score and prevented the thinning of the inner retinal layer while therapeutic treatment was not protective on visual outcomes. CONCLUSIONS: A prophylactic LA treatment is necessary to protect from visual loss and retinal thinning in EAE-ON, suggesting that a clinical ON trial starting therapy after the onset of symptoms may not be successful.

Proteomic Profile of Carbonylated Proteins Screen Regulation of Apoptosis via CaMK Signaling in Response to Regular Aerobic Exercise.

To research carbonylated proteins and screen molecular targets in the rat striatum on regular aerobic exercise, male Sprague-Dawley rats (13 months old, n = 24) were randomly divided into middle-aged sedentary control (M-SED) and aerobic exercise (M-EX) groups (n = 12 each). Maximum oxygen consumption (VO2max) gradually increased from 50%-55% to 65%-70% for a total of 10 weeks. A total of 36 carbonylated proteins with modified oxidative sites were identified by Electrospray Ionization-Quadrupole-Time of Flight-Mass Spectrometer (ESI-Q-TOF-MS), including 17 carbonylated proteins unique to the M-SED group, calcium/calmodulin-dependent protein kinase type II subunit beta (CaMKIIß), and heterogeneous nuclear ribonucleoprotein A2/B1 (Hnrnpa2b1), among others, and 19 specific to the M-EX group, ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCH-L1), and malic enzyme, among others. Regular aerobic exercise improved behavioral and stereological indicators, promoted normal apoptosis (P < 0.01), alleviated carbonylation of the CaMKIIß and Hnrnpa2b1, but induced carbonylation of the UCH-L1, and significantly upregulated the expression levels of CaMKIIß, CaMKIIα, and Vdac1 (p < 0.01) and Hnrnpa2b1 and UCH-L1 (p < 0.01), as well as the phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin pathways (PI3K/Akt/mTOR) pathway-related genes Akt and mTOR. Regular aerobic exercise for 10 weeks (incremental for the first 6 weeks followed by constant loading for 4 weeks) enhanced carbonylation of CaMKIIß, Hnrnpa2b1, and modulated apoptosis via activation of CaMK and phosphoinositide 3-kinase/protein kinase B/mTOR signaling. It also promoted normal apoptosis in the rat striatum, which may have protective effects in neurons.

Mechanism of Protein Carbonylation in Glutathione-Depleted Rat Brain Slices.

This study was conducted to further our understanding about the link between lipid peroxidation and protein carbonylation in rat brain slices incubated with the glutathione (GSH)-depletor diethyl maleate. Using this in vitro system of oxidative stress, we found that there is a significant lag between the appearance of carbonylated proteins and GSH depletion, which seems to be due to the removal of oxidized species early on in the incubation by the mitochondrial Lon protease. Upon acute GSH depletion, protein carbonyls accumulated mostly in mitochondria and to a lesser degree in other subcellular fractions that also contain high levels of polyunsaturated lipids. This result is consistent with our previous findings suggesting that lipid hydroperoxides mediate the oxidation of proteins in this system. However, these lipid hydroperoxides are not produced by oxidation of free arachidonic acid or other polyunsaturated free fatty acids by lipooxygenases or cyclooxygenases. Finally, γ-glutamyl semialdehyde and 2-amino-adipic semialdehyde were identified by HPLC as the carbonyl-containing amino acid residues, indicating that proteins are carbonylated by metal ion-catalyzed oxidation of lysine, arginine and proline residues. The present findings are important in the context of neurological disorders that exhibit increased lipid peroxidation and protein carbonylation, such as Parkinson's disease, Alzheimer's disease, and multiple sclerosis.

[Oxidative carbonylation of proteins in experimental hind-limb ischaemia-reperfusion injury]. / Okislitel'noe karbonilirovanie belkov pri éksperimental'noi ishemii i reperfuzii nizhnikh konechnostei.

Formation of carbonylated protein derivatives is one of the key signalling pathways in cellular damage and may be regarded as a reliable marker in cellular injury. It allows evaluating both direct effects of reactive oxygen species and indirect interrelations with the secondary by-products of oxidation. The present study was undertaken to investigate the activity of lysosomal cysteine proteinases (cathepsins B and L) in blood serum and the arterial wall in an experimental model of ischaemia and ischaemia-reperfusion injury. To this was added comprehensive assessment of oxidative modification of proteins derived from blood serum and the arterial wall, namely, calculating the area under the curve of the absorption spectrum of the products of protein carbonylation, the proportion of the primary and secondary markers of oxidative stress, as well as the reserve and adaptation potential. The obtained findings were indicative of the development of oxidative stress in the model of ischaemia-reperfusion injury from day 1 to day 7, and in the ischaemia model on day 3 and day 5 in both the vascular wall and blood serum, which was accompanied by activation of cathepsins B and L. Reversible oxidation of proteins was observed on days 3 and 5 in the experimental ischaemia model and on days 1 and 7 in the ischaemia-reperfusion injury model, which was confirmed by the predominance of the primary markers of oxidative stress. Irreversible oxidation of proteins, i. e., the predominance of the secondary markers, was suggestive of the enhancement of oxidative stress, its transition to the late stage, leading to the loss of biological properties of proteins and eventually followed by their aggregation and degradation as seen in the ischaemia-reperfusion injury model on days 3 and 5. Analysing the obtained findings revealed direct correlation between the total area under the curve of oxidative modification of proteins and overall activity of cathepsin L in the ischaemia model: in blood serum on days 3 and 5, in the vascular wall for cathepsins B and L on day 5; in the ischaemia-reperfusion injury model: the activity of cathepsin L in blood serum on day 3, in the vascular wall on day 5 for cathepsins B and L.

Cerebral Oxidative Stress and Microvasculature Defects in TNF-α Expressing Transgenic and Porphyromonas gingivalis-Infected ApoE-/- Mice.

The polymicrobial dysbiotic subgingival biofilm microbes associated with periodontal disease appear to contribute to developing pathologies in distal body sites, including the brain. This study examined oxidative stress, in the form of increased protein carbonylation and oxidative protein damage, in the tumor necrosis factor-α (TNF-α) transgenic mouse that models inflammatory TNF-α excess during bacterial infection; and in the apolipoprotein knockout (ApoE-/-) mouse brains, following Porphyromonas gingivalis gingival monoinfection. Following 2,4-dinitrophenylhydrazine derivatization, carbonyl groups were detected in frontal lobe brain tissue lysates by immunoblotting and immunohistochemical analysis of fixed tissue sections from the frontotemporal lobe and the hippocampus. Immunoblot analysis confirmed the presence of variable carbonyl content and oxidative protein damage in all lysates, with TNF-α transgenic blots exhibiting increased protein carbonyl content, with consistently prominent bands at 25 kDa (p = 0.0001), 43 kDa, and 68 kDa, over wild-type mice. Compared to sham-infected ApoE-/- mouse blots, P. gingivalis-infected brain tissue blots demonstrated the greatest detectable protein carbonyl content overall, with numerous prominent bands at 25 kDa (p = 0.001) and 43 kDa (p = 0.0001) and an exclusive band to this group between 30-43 kDa* (p = 0.0001). In addition, marked immunostaining was detected exclusively in the microvasculature in P. gingivalis-infected hippocampal tissue sections, compared to sham-infected, wild-type, and TNF-α transgenic mice. This study revealed that the hippocampal microvascular structure of P. gingivalis-infected ApoE-/- mice possesses elevated oxidative stress levels, resulting in the associated tight junction proteins being susceptible to increased oxidative/proteolytic degradation, leading to a loss of functional integrity.

Assessing the risk of cytomegalovirus DNAaemia in allogeneic stem cell transplant recipients by monitoring oxidative-stress markers in plasma.

The level of antioxidants, such as thiol-containing tripeptide glutathione (GSH), in cytomegalovirus (CMV)-infected cells is notably increased. We previously showed that GSH levels in plasma, as measured by untargeted 1H nuclear magnetic resonance, are higher in allogeneic stem cell transplant (allo-SCT) recipients who subsequently develop CMV viraemia. We hypothesized that the net level of oxidative-stress markers present in plasma may be reduced in patients who develop CMV DNAaemia compared to those who do not. We serially monitored the levels of malondialdehyde (MDA) and carbonylated proteins (CPs) early after allo-SCT and assessed whether they could predict the occurrence of CMV DNAaemia. MDA levels were measured in 43 patients (28 had CMV DNAaemia) and CPs were quantified in 53 patients (38 patients developed CMV DNAaemia). The area under the curve (AUC) value for MDA, but not for CPs, was significantly lower in patients who subsequently developed CMV DNAaemia compared to those who remained DNAaemia-free (P=0.043). A trend toward lower MDA AUC values was observed in episodes of CMV DNAaemia with faster CMV replicative kinetics and in those who reached higher peak CMV DNA levels. Moreover, receiver operating characteristic curve analyses indicated that the MDA biomarker had the predictive ability to discriminate between patients with or without subsequent CMV DNAaemia (AUC=0.69, 95 % confidence interval 0.51-0.85, P=0.05). In summary, serial quantitation of MDA may be useful for individualizing antiviral prophylaxis therapies (targeted prophylaxis) in the upcoming era of new antiviral drugs with improved safety profiles.

Obesity, bariatric surgery and oxidative stress.

INTRODUCTION:: Obesity refers to the accumulation of fatty tissues and it favors the occurrence of oxidative stress. Alternatives that can contribute to body weight reduction have been investigated in order to reduce the production of reactive oxygen species responsible for tissue damage. The aim of the current study was to assess whether the oxidant and antioxidant markers of obese women before and after bariatric surgery were able to reduce oxidative damage. METHOD:: We have assessed 16 morbidly obese women five days before and 180 days after the surgery. The control group comprised 16 non-obese women. Levels of thiobarbituric acid-reactive substances, carbonylated proteins, reduced glutathione and ascorbic acid were assessed in the patients' plasma. RESULTS:: Levels of lipid peroxidation and protein carbonylation in the pre-surgical obese women were higher than those of the controls and post-surgical obese women. Levels of reduced glutathione in the pre-surgical obese women were high compared to the controls, and declined after surgery. Levels of ascorbic acid fell in the pre--surgical obese women compared to the control and post-surgical obese women. CONCLUSION:: Body weight influences the production of reactive oxygen species. Bariatric surgery, combined with weight loss and vitamin supplementation, reduces cellular oxidation, thus reducing tissue damage.

The ET-1-mediated carbonylation and degradation of ANXA1 induce inflammatory phenotype and proliferation of pulmonary artery smooth muscle cells in HPS.

Hepatopulmonary syndrome (HPS) is a serious complication of advanced liver disease, which markedly increases mortality. Pulmonary vascular remodelling (PVR) induced by circulating mediators plays an important role in the pathogenesis of HPS, while the underlying mechanism remains undefined. In the present study, we reported that endothelin-1 (ET-1) is up-regulated and annexin A1(ANXA1) is down-regulated in HPS rat, and ET-1 decreases the ANXA1 expression in a dose-dependent manner in rat pulmonary arterial smooth muscle cells (PASMCs). Then, we showed that ANXA1 can decrease nuclear p-ERK1/2 accumulation and decrease the cyclin D1 expression, thus resulting in the subsequent inhibition of PASMCs proliferation. As previously reported, we confirmed that ET-1 decreases the ANXA1 protein levels by the carbonylation and degradation of ANXA1. In conclusion, our research links the signaling cascade of ET1-ANXA1-cell proliferation to a potential therapeutic strategy for blocking IPS-associated PVR.

Obesity, bariatric surgery and oxidative stress / Obesidade, cirurgia bariátrica e estresse oxidativo

Summary Introduction: Obesity refers to the accumulation of fatty tissues and it favors the occurrence of oxidative stress. Alternatives that can contribute to body weight reduction have been investigated in order to reduce the production of reactive oxygen species responsible for tissue damage. The aim of the current study was to assess whether the oxidant and antioxidant markers of obese women before and after bariatric surgery were able to reduce oxidative damage. Method: We have assessed 16 morbidly obese women five days before and 180 days after the surgery. The control group comprised 16 non-obese women. Levels of thiobarbituric acid-reactive substances, carbonylated proteins, reduced glutathione and ascorbic acid were assessed in the patients' plasma. Results: Levels of lipid peroxidation and protein carbonylation in the pre-surgical obese women were higher than those of the controls and post-surgical obese women. Levels of reduced glutathione in the pre-surgical obese women were high compared to the controls, and declined after surgery. Levels of ascorbic acid fell in the pre--surgical obese women compared to the control and post-surgical obese women. Conclusion: Body weight influences the production of reactive oxygen species. Bariatric surgery, combined with weight loss and vitamin supplementation, reduces cellular oxidation, thus reducing tissue damage.

Resumo Introdução: Na obesidade, verifica-se um acúmulo de tecido adiposo, o que favorece a ocorrência de estresse oxidativo. A fim de diminuir a produção das espécies reativas que levam a danos teciduais, buscam-se alternativas que contribuam para a redução do peso corporal. Este estudo avaliou se os marcadores oxidantes e antioxidantes de obesas antes e após cirurgia bariátrica reduziram o dano oxidativo. Método: Foram avaliadas 16 mulheres obesas mórbidas cinco dias antes e 180 dias após o procedimento cirúrgico. O grupo controle constituiu-se de 16 mulheres não obesas. Os níveis das substâncias reativas ao ácido tiobarbitúrico, das proteínas carboniladas, da glutationa reduzida e do ácido ascórbico foram avaliados no plasma dessas pacientes. Resultados: Os níveis de lipoperoxidação e da carbonilação de proteínas nas obesas pré-cirúrgicas eram mais elevados quando comparados ao controle e às obesas pós-cirúrgicas; os níveis de glutationa reduzida eram maiores nas obesas pré-cirúrgicas em comparação ao controle e diminuíram após a cirurgia; os níveis de ácido ascórbico eram menores nas obesas pré-cirúrgicas em relação ao controle e às obesas pós-cirúrgicas. Conclusão: Observou-se que a massa corporal influenciou na produção das espécies reativas. A cirurgia bariátrica, somada à perda de peso e à suplementação vitamínica, diminui a oxidação celular e, com isso, reduz os danos teciduais.

Variations in Oxidative Stress Levels in 3 Days Follow-up in Ultramarathon Mountain Race Athletes.

Spanidis, Y, Stagos, D, Orfanou, M, Goutzourelas, N, Bar-or, D, Spandidos, D, and Kouretas, D. Variations in oxidative stress levels in 3 days follow-up in ultramarathon mountain race athletes. J Strength Cond Res 31(3): 582-594, 2017-The aim of the present study was the monitoring of the redox status of runners participating in a mountain ultramarathon race of 103 km. Blood samples from 12 runners were collected prerace and 24, 48, and 72 hours postrace. The samples were analyzed by using conventional oxidative stress markers, such as protein carbonyls (CARB), thiobarbituric acid reactive substances (TBARS), total antioxidant capacity (TAC) in plasma, as well as glutathione (GSH) levels and catalase (CAT) activity in erythrocytes. In addition, 2 novel markers, the static oxidation-reduction potential marker (sORP) and the capacity oxidation-reduction potential (cORP), were measured in plasma. The results showed significant increase in sORP levels and significant decrease in cORP and GSH levels postrace compared with prerace. The other markers did not exhibit significant changes postrace compared with prerace. Furthermore, an interindividual analysis showed that in all athletes but one sORP was increased, whereas cORP was decreased. Moreover, GSH levels were decreased in all athletes at least at 2 time points postrace compared with prerace. The other markers exhibited great variations between different athletes. In conclusion, ORP and GSH markers suggested that oxidative stress has existed even 3 days post ultramarathon race. The practical applications from these results would be that the most effective markers for short-term monitoring of ultramarathon mountain race-induced oxidative stress were sORP, cORP, and GSH. Also, administration of supplements enhancing especially GSH is recommended during ultramarathon mountain races to prevent manifestation of pathological conditions.

Protein carbonyl content: a novel biomarker for aging in HIV/AIDS patients

Abstract Background: The major complications of “treated” Human Immunodeficiency Virus (HIV) infection are cardiovascular disease, malignancy, renal disease, liver disease, bone disease, and perhaps neurological complications, which are phenomena of the normal aging process occurring at an earlier age in the HIV-infected population. The present study is aimed to explore protein carbonyl content as a biomarker for detecting oxidative DNA damage induced ART toxicity and/or accelerated aging in HIV/AIDS patients. Objective: To investigate the potential of carbonyl content as a biomarker for detecting oxidative Deoxyribonucleic acid (DNA) damage induced Antiretroviral Theraphy (ART) toxicity and/or accelerated aging in HIV/AIDS patients. Methods: In this case–control study a total 600 subjects were included. All subjects were randomly selected and grouped as HIV-negative (control group) (n = 300), HIV-infected ART naive (n = 100), HIV-infected on first line ART (n = 100), and HIV-infected on second line ART (n = 100). Seronegative control subjects were age- and sex-matched with the ART naive patients and the two other groups. Carbonyl protein was determined by the method described in Levine et al. DNA damage marker 8-OH-dG was determined using 8-hydroxy-2-deoxy Guanosine StressXpress ELA Kit by StressMarq Biosciences. Results: Protein carbonyl content levels and oxidative DNA damage were significantly higher (p < 0.05) in HIV-infected patients on second line ART and HIV-infected patients on first line ART than ART naive patients and controls. In a linear regression analysis, increased protein carbonyl content was positively associated with increased DNA damage (OR: 0.356; 95% CI: 0.287–0.426) p < 0.05. Conclusions: Carbonyl content may has a role as a biomarker for detecting oxidative DNA damage induced ART toxicity and/or accelerated aging in HIV/AIDS patients. Larger studies are warranted to elucidate the role of carbonyl content as a biomarker for premature aging in HIV/AIDS patients.